Mixing time is one of the most important characteristics of bioreactors used to validate computational fluid dynamics (CFD) models in bioprocess. The pH tracer based method is commonly used to estimate mixing time by monitoring the pH change after perturbation to the system. However, pH probe lag is known to introduce errors in the test results. To determine the impact of probe lag on the measurement, pH probe response times were investigated. A second order mathematical model was established to describe the probe response profile and calculate the probe transfer function which was used to correct the empirical results. The corrected mixing time was used to validate the CFD models and also showed that the impact of pH probe response time on mixing time measurement is significant even in larger (pilot and manufacturing) scales. In addition, comparison with a conductance based tracer method is also discussed in this paper. (C) 2014 Elsevier Ltd. All rights reserved.